Today, there is an increasing demand for fluorescent polymers in very diverse sectors. Thus, in the field of security, fluorescent marking is of interest for fighting against counterfeiting [1]. In the field of forensics, it is applied in police investigations [2]. Fluorescent polymers are also used in chemical sensors for detecting pollutants or toxic compounds in water or in air [3] [4] [5] and [6].
Fluorescent polymers may also find applications in the field of imaging, notably confocal microscopy [7] or fluorescence microscopy.
In the therapeutical or pharmaceutical fields, fluorescent polymers may be coupled with a biological ligand and thus used in tests for detecting target molecules [8].
In the field of graphic arts, they may be used in paints, inks for textile printing or for engraving [9].
Other original applications of fluorescent polymers are also listed in the literature, such as the one described by Uchiyama et al. [10] who have developed fluorescent molecular thermometers by coupling poly(N-isopropylacrylamide), known for undergoing a phase transition in an aqueous solution around 32° C., with a fluorescent derivative of the benzofurazane type, the fluorescence of which depends on the polarity of the solvent.
If the interest is now focused on coumarins, umbelliferone (or 7-hydroxycoumarin) has blue fluorescence emission (300, 305 and 325 nm) when it is excited by a radiation located in the ultraviolet range. A great number of its derivatives are of great importance in physics, chemistry, biology and medicine and have been used for various applications [11].
In particular, the photochemical and photophysical properties in the excited state of 4-methylumbelliferone (4MU) have been known for a long time and this fluorophore was used as a pH probe and as a laser dye [12] [13] and [14].
In the biomedical field, 4MU has also proved to be an inhibitor of the synthesis of hyaluronic acid [16] [17] [18], which is one of the main components of the extracellular matrix which significantly contributes to proliferation and migration of cells and which is thus involved in the progression of certain malignant tumors. A recent review by Yu et al. [19] moreover describes the study of more than 150 derivatives of the coumarin type and their efficiency as anti-HIV agents. Lipophilic 4-heptadecyl-7-hydroxycoumarin, as for it, has been used as a probe for studying properties of phospholipid bilayers at the water/lipid interface or for measuring pH differences at membrane interfaces [20].
Easy to couple with other entities through conventional chemistry, coumarins are attractive because of their original properties, i.e. notably absorption and emission of light which may be modulated, and reversible photodimerization. The idea of using them in polymers for synthesizing macromolecular architectures with specific properties, therefore appeared rapidly [11].
For example, the photodimerization and photocleavage properties of coumarins have been utilized for making liquid crystal polymers and biodegradable polypeptides. Other uses have the purpose of obtaining electroluminescent polymers, or further for collecting and transferring solar energy.
In order to obtain fluorescent polymers, there exist several methods. The simplest one consists of dispersing fluorescent molecules in a matrix of the polymer type. Campbell and Bartlett [7] have thereby synthesized monodispersed poly(methylmethacrylate) (PMMA) spheres by copolymerization of methyl methacrylate and of methacrylic acid in the presence of a non-polymerizable dye. Strongly fluorescent PMMA colloids were thus obtained.
Molecular Probes® [21] markets polystyrene microspheres with various sizes and different colors depending on the nature of the dye which is dispersed therein (FluoSpheres®). These fluorescent beads have been used as microinjectable cell tracers, as antigen markers, for flow cytometry or further for studying phagocytosis phenomena or measuring blood flow in tissues. Although these microspheres have high gloss and do not seem to undergo any notable photobleaching, the fact that the dye is not covalently attached to the matrix implies an heterophase formation and a risk of salting out the dye into the biological medium. Covalent grafting of the fluorophore is therefore preferable in order to avoid these drawbacks.
Another listed method therefore consists of post-functionalizing  non-fluorescent polymer chains from substituents allowing reactive coupling with a fluorophore. Saegusa [22] thus described the synthesis of poly-oxazolines by polymerization of 2-methyl-2-oxazoline by ring opening, and then the functionalization of the polymer by hydrolysis of the side methyl group, followed by coupling with 7-coumaryloxyacetic acid.
Also, the document of Rhum and Matthews [23] relates to the copolymerization of hydroxyethyl methacrylate (HEMA) and of methyl methacrylate (MMA), followed by the coupling of the hydroxyl group of HEMA with 4-carboxymethyl-umbelliferone, thereby giving a pH indicator which is not water-soluble.
Similarly, the document of Bouma and Celebuski [24] is related to derivatives of 7-hydroxycoumarin having substituents in position 4. These substituents include functional groups allowing them to be coupled with biological molecules, these are typically substituents with —OH, —SH or —NH2 end groups.
However, this post-functionalization method is only applied to a restricted number of polymers having adequate functional groups such as —COOH, —OH, —SH, —NH2, or —NCO and does not allow control either of the level or of the distribution of the grafting of the fluorophore on the polymer.
It should be noted that these functional groups are not groups which may be described as polymerizable groups.
The fluorophores which may be used directly as a monomer or comonomer, have the advantage of eliminating the disadvantages mentioned above. Pitschke et al. [25] describe the synthesis of a monomer derived from 7-aminocoumarin, substituted in position 3 with a polymerizable styrenic group (Formula 1a below). However, substitution of the coumarin ring in position 3 is known for deeply changing the electronic properties of the original coumarin ring [23].
Rathbone et al. [26], as for them, describe the use of the 7-hydroxy—4-methylcoumarin acrylate monomer (Formula 1b below) for the synthesis of fluorescent polymers having molecular print. However the blocking of the phenol function in position 7 by the acrylate group causes the loss of an interaction site via a hydrogen bond essential for many applications such as notably pH measurements.
Structure of the fluorescent monomers described by Pitschke et al. (1a) and by Rathbone et al. (1b).
As a summary, the dispersion of dye in polymers is not satisfactory since in this case the dye is not covalently grafted to the polymer, by which it is not possible to ensure sufficient durability and this causes a risk of salting-out of the dye.
Moreover, the reactive coupling of a dye on preformed polymer chains, does not allow control of the grafting (level and distribution) and therefore of the physicochemical properties of the materials on the one hand and restricts both the number of polymers and the type of dye which may be used, to compounds including reactive functions such as —OH, —NH2, —SH, —NCO, —COOH on the other hand.
Finally, we noticed that only a restricted number of directly polymerizable fluorophores are marketed to this day and the rare coumarin derivatives chemically modified so as to become polymerizable, no longer have the original properties of coumarin.
Therefore, considering the foregoing, there exists a need for a fluorescent compound or fluorophore derived from coumarin and notably from 7-hydroxycoumarin which is directly polymerizable i.e. provided with a polymerizable substituent, but in which the electronic properties and the photoluminescence of the coumarin ring are not or substantially not affected by this polymerizable substituent.
In other words, there exists a need for a polymerizable fluorescent compound derived from coumarin as a basic fluorophore in which the original properties, notably the electronic properties and photoluminescence properties of this basic fluorophore are entirely or at least for a major part preserved.
In particular, it would be interesting to be able to have a polymerizable monomer, compound derived from 7-hydroxycoumarin in which all the properties of 7-hydroxycoumarin would be preserved and in which the phenol function in position 7 of the coumarin ring, the importance of which is known in many applications, would be left intact.
Further, there exists a need for such a polymerizable fluorescent compound which may be easily polymerized, notably by radical polymerization methods either controlled or not, and which may therefore be covalently bound to chains of homopolymers and/or copolymers for example of vinyl, acrylic or styrenic homopolymers and/or copolymers.
The goal of the invention is to provide a polymerizable compound, further called a polymerizable monomer, which inter alia meets the needs listed above.
The goal of the invention is further to provide a polymerizable compound which does not have the drawbacks, defects, limitations and disadvantages of the polymerizable compounds mentioned above and which solves the problems of these compounds.